Perfluoroalkyl substances (pfas) phytoremediation by manipulating soil properties and plant management

ABSTRACT

Phytoremediation processes, methods, materials and compositions to remediate soil, sediment and groundwater that is contaminated by per- and polyfluoroalkyl substances (PFAS) via phytoextraction which includes the uptake and translocation of contaminants in the contaminated media by plant roots into the above ground portions of the plants. The plants can be selected from sixteen plants as well as other plants and the invention can include managing soil salinity levels of the plants, manipulating amounts of organic matter in the contaminated site media, managing pH levels of the contaminated sites, utilizing double cropping systems, utilizing double-canopy system, and managing harvest methodology of the plants.

RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent applicationSer. No. 16/793,103 filed Feb. 18, 2020, now allowed, which claims thebenefit of priority to U.S. Provisional Patent Application Ser. No.62/847,634 filed May 14, 2019. The entire disclosure of each of theapplications listed in this paragraph are incorporated herein byspecific reference thereto.

FIELD OF INVENTION

This invention relates to phytoremediation, and in particular tophytoremediation processes, methods, materials and compositions toremediate soil, sediment and groundwater that is contaminated by per-and polyfluoroalkyl substances (PFAS) via phytoextraction which includesthe uptake and translocation of contaminants in the soil by plant rootsinto the above ground portions of the plants.

BACKGROUND AND PRIOR ART

PFAS are a large group of synthetic compounds that have been broadlyused to make various products more resistant to stains, grease, andwater since the 1940s and there is mounting evidence that exposure abovespecific levels to certain PFAS leads to adverse health effects.Examples of products with which PFAS have been used extensively includenonstick cookware, stain resistant textiles, and waterproof clothing.They have also been used in many food packaging and firefightingmaterials. Because they help reduce friction, they are also used in avariety of other industries, including aerospace, automotive, buildingand construction, and electronics. PFAS are known to break down slowlyin the environment and are characterized as persistent. Due to theirpervasive use and persistence, there is widespread human exposure toPFAS including perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS).

There is a need for a viable and cost-effective phytoremediation cleanupmethodology to address PFAS impacted soil, sediments, and groundwater.The methodology would be all the more desirable if it met core elementsoutlined in the American Society of Testing Materials (ASTM) E2893Standard Guide for Greener Cleanups that include minimizing greenhousegas emissions, air pollutants, use of materials, generation of waste,disturbance to land and ecosystems, and noise and light disturbance.

Current approaches to cleanup of impacted soil and sediment consists ofexpensive and energy consumptive excavation and removal.

In November 2016 the US EPA issued health advisories for PFOA and PFOS,based on the agency's assessment of the latest peer-reviewed science, toprovide drinking water system operators, and state, tribal, and localofficials who have the primary responsibility for overseeing thesesystems, with information on the health risks of these chemicals. Withthe issuance of these advisories greater focus has been brought toassessing the fate and potential remediation of PFAS in the environment.In February 2019, EPA issued a PFAS Action Plan to further understandand reduce PFAS risks to the public. The Action Plan describes EPA'sapproaches to addressing current PFAS contamination and specificallyincludes steps to propose designating PFOA and PFOS as “hazardoussubstances”. In addition, the plan includes developing toxicity valuesfor perfluorobutane sulfonic acid (PFBS) and developing guidance forcleanup actions where groundwater is contaminated with PFOA and PFOS.

Studies have indicated that PFAS accumulates in plants where it is foundin the soil. However, these studies have not addressed processes,methods, materials and/or compositions to effectuate greater plantuptake for the purpose of phytoremediation.

Prospects of a phytoremediation approach for cleaning up PFAS-impactedsoil and sediments were evaluated in a recent study from Europeanresearchers who found various plants species to be promising candidatesfor phytoremediation of PFAS (Gobelius, L., J. Lewis, and I. Ahrens,2017. Plant Uptake of Per- and Polyfluoroalkyl Substances at aContaminated Fire Training Facility to Evaluate the PhytoremediationPotential of Various Plant Species. Environ. Sci. Technol. 2017, 51,12602-12610). This research consisted of a survey of plants present at asite known to be contaminated by PFAS.

Other related research has investigated the presence of PFAS inagricultural crops, largely to assess potential threats posed by PFAS infood supplies. These studies showed agricultural crop plantsaccumulating PFAS compounds in both root and above-ground tissue at lowlevels (Navarro et al 2017, Ghisi et al. 2018). The amount ofaccumulation depends on a variety of factors including plant species(Navarro et al. 2017, Ghisi et al. 2018), PFAS group and chain length(Blaine et al. 2014, Ghisi et al. 2018), water or soil concentration(Blaine et al. 2014, Ghisi et al. 2018), organic carbon content of thesoil (Blaine et al. 2014), salinity and pH (Zhao et al. 2013). Morerecently, Zhang et al. (2019) reported on the uptake and accumulation ofseven PFAS compounds by the wetland species Juncus effuses. Theyreported removal efficiencies from solution as high as 11.4% for spikedPFAS, but reported little translocation to above-ground components ofthe plant.

These previous studies differ from the invention presented hereinbecause our invention focused on maximizing above-ground plantaccumulation and was developed via a replicated statistically-basedrandomized block design study within an environmentally controlledgreenhouse that produced and demonstrated repeatable results forming thebasis of our phytoremediation processes, methods, materials andcompositions which remediate soil, sediment, and groundwatercontaminated by per- and polyfluoroalkyl substances (PFAS) viaphytoextraction.

SUMMARY OF THE INVENTION

A primary objective of the present invention is to providephytoremediation processes, methods, materials, and compositions toremediate soil that is contaminated by per- and polyfluoroalkylsubstances (PFAS).

A secondary objective of the present invention is to providephytoremediation processes, methods, materials, and compositions toremediate sediment that is contaminated by per- and polyfluoroalkylsubstances (PFAS).

A third objective of the present invention it to providephytoremediation processes, methods, materials, and compositions toremediate groundwater that is contaminated by per- and polyfluoroalkylsubstances (PFAS).

A fourth objective of the present invention is to maximizephytoremediation of per- and polyfluoroalkyl substances (PFAS) atcontaminated sites with selected plants by managing soil salinity levelsof the plants, manipulating amounts of organic matter in thecontaminated media, managing pH levels of the contaminated media, andmanaging harvest methodology of the plants.

A fifth objective of the present invention is to maximizephytoremediation of per- and polyfluoroalkyl substances (PFAS) atcontaminated sites by utilizing coordinated double cropping systems tooverlap cool season and warm season plants and utilizing coordinateddual cropping systems with shade tolerant understory forbs and grasseswith tree species.

A method for increasing the amount of per- and polyfluoroalkylsubstances (PFAS) that a plant will accumulate from PFAS contaminatedsoil, sediment, and groundwater, can include the steps of growing liveselected plants in per- and polyfluoroalkyl substances (PFAS)contaminated soil, sediments, or groundwater, and providing forphytoremediation via phytoextraction of the per- and polyfluoroalkylsubstances (PFAS) from the contaminated media.

The providing step further includes the steps of managing soil salinitylevels, manipulating amounts of organic matter in the contaminatedmedia, using a double-cropping system, using a double-canopy system,managing pH levels of the contaminated sites and managing harvestmethodology of the plants.

The live selected plant can be selected from any one of Amaranthustricolor, Betula nigra, Brassica juncea, Cynodon dactylon, Esquisetumhyemale, Schedonorus arundinaceus, Festuca rubra (Red Fescue) and itssubspecies, Helianthus annuus, Liquidambar styraciflua, Liriodendrontulipifera, Trifolium incamatum, Platanus occidentalis, and Salix nigra.

The live selected plant can be Festuca rubra (Red Fescue) and itssubspecies.

The live selected plant can be Liquidambar styraciflua (Sweetgum-LP).

The live selected plant can be Salix nigra (Black willow-LP) leaves andpetioles.

The step of managing soil salinity levels of the plants can include thesteps of increasing soil salinity by addition of salt, othersalinity-increasing soil amendments and irrigants within the range ofplant tolerances.

The step of manipulating amounts of organic matter in the contaminatedsites, can include the step of reducing the amounts of organic matterand soluble carbon levels in the contaminated sites.

The step of reducing the amounts of the organic matter and solublecarbon levels in the contaminated sites, can include the steps of atleast one of tilling the contaminated soil and adding inorganic nitrogento the contaminated soil.

The step of managing pH levels of the contaminated sites can include thesteps of providing soil and soil-water in the contaminated media to becircumneutral (7.0 Standard Units (+/−0.5)).

The step of managing harvest methodology of the plant, can includes thesteps of providing more frequent and timed harvests of the selectedplant at earlier stages of growth when protein contents are greater.

The per- and polyfluoroalkyl substances (PFAS) can include the compoundsof perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), andperfluorobutane sulfonic acid (PFBS).

The per- and polyfluoroalkyl substances (PFAS) can include the compoundsof perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS),perfluorobutane sulfonic acid (PFBS), tridecafluorohexane-1-sulfonicacid potassium salt (PFHxS), perfluoropentanoic acid (PFPeA), andundecafluorohexanoic acid (PFHxA).

The providing step can include the steps of removing significantportions of compounds of per- and polyfluoroalkyl substances (PFAS)through phytoextraction and harvesting of above ground plant tissue.

The contaminated site can be selected from at least one of PFAScontaminated soil, PFAS contaminated sediment, and PFAS contaminatedgroundwater.

A process for hyperaccumulating multiple per- and polyfluoroalkylsubstances PFAS compounds, can include the steps of growing liveselected plants in per- and polyfluoroalkyl substances (PFAS)contaminated soil, sediments, or groundwater, the live plants selectedfrom Amaranthus tricolor, Betula nigra, Brassica juncea, Cynodondactylon, Esquisetum hyemale, Schedonorus arundinaceus, Festuca rubra(and its subspecies), Helianthus annuus, Liquidambar styraciflua,Liriodendron tulipifera, Trifolium incamatum, Platanus occidentalis, andSalix nigra, and phytoremediating via phytoextraction of the per- andpolyfluoroalkyl substances (PFAS) from the contaminated media, the PFAScontaminants including the compounds perfluorooctanoic acid (PFOA),perfluorooctane sulfonate (PFOS), and perfluorobutane sulfonic acid(PFBS).

A process for hyperaccumulating multiple per- and polyfluoroalkylsubstances PFAS compounds, can include the steps of growing liveselected plants in per- and polyfluoroalkyl substances (PFAS)contaminated soil, sediments, or groundwater, the live plants selectedfrom Amaranthus tricolor, Betula nigra, Brassica juncea, Cynodondactylon, Esquisetum hyemale, Schedonorus arundinaceus, Festuca rubra(and its subspecies), Helianthus annuus, Liquidambar styraciflua,Liriodendron tulipifera, Trifolium incamatum, and Platanus occidentalis,and Salix nigra, and phytoremediating via phytoextraction of the per-and polyfluoroalkyl substances (PFAS) from the contaminated media, thePFAS contaminants including the compounds perfluorooctanoic acid (PFOA),perfluorooctane sulfonate (PFOS), perfluorobutane sulfonic acid (PFBS),tridecafluorohexane-1-sulfonic acid potassium salt (PFHxS),perfluoropentanoic acid (PFPeA), and undecafluorohexanoic acid (PFHxA).

Further objectives and advantages of this invention will be apparentfrom the following detailed description of the presently preferredembodiments which are illustrated schematically in the accompanyingdrawings.

BRIEF DESCRIPTION OF THE FIGURES

The drawing figures depict one or more implementations in accord withthe present concepts, by way of example only, not by way of limitations.In the figures, like reference numerals refer to the same or similarelements.

FIG. 1 is a flow chart summary of the phytoremediation processes,methods, materials and compositions to remediate soil, sediment, andgroundwater that is contaminated by per- and polyfluoroalkyl substances(PFAS) via phytoextraction which includes the uptake and translocationof contaminants in the soil, sediment, and groundwater by plant rootsinto the above ground portions of the plants.

FIG. 2 are bar graphs of the Bioconcentration Factors (BCFs) of Festucarubra (Red Fescue) for six target PFAS compounds after 24 days ofinitial dosing with PFAS contaminant solution.

FIG. 3 are bar graphs of the BCFs of Festuca rubra (Red Fescue) for sixtarget PFAS compounds after 98 days of initial dosing with PFAScontaminant solution.

FIG. 4 are bar graphs of the BCFs of Cynodon dactylon (Bermudagrass) forsix target PFAS compounds after 24 days of initial dosing with PFAScontaminant solution.

FIG. 5 are bar graphs of the BCFs of Cynodon dactylon (Bermudagrass) forsix target PFAS compounds after 98 days of initial dosing with PFAScontaminant solution.

FIG. 6 are bar graphs of the BCFs of Liquidambar styraciflua (Sweetgum)for six target PFAS compounds after 18 days of initial dosing with PFAScontaminant solution.

FIG. 7 are bar graphs of the BCFs of Liquidambar styraciflua (Sweetgum)for six target PFAS compounds after 92 days of initial dosing with PFAScontaminant solution.

FIG. 8 are bar graphs of the BCFs of Salix nigra (Black Willow) for sixtarget PFAS compounds after 18 days of initial dosing with PFAScontaminant solution.

FIG. 9 are bar graphs of the BCFs of Salix nigra (Black Willow) for sixtarget PFAS compounds after 92 days of initial dosing with PFAScontaminant solution.

FIG. 10 are bar graphs of the BCFs of Trifolium incamatum (CrimsonClover) for six target PFAS compounds after 24 days of initial dosingwith PFAS contaminant solution.

FIG. 11 are bar graphs of the BCFs of Schedonorus arundinaceus (TallFescue) for six target PFAS compounds after 24 days of initial dosingwith PFAS contaminant solution.

FIG. 12 are bar graphs of the BCFs of Schedonorus arundinaceus (TallFescue) for six target PFAS compounds after 98 days of initial dosingwith PFAS contaminant solution.

FIG. 13 are bar graphs of the BCFs of Brassica juncea (Mustard) for sixtarget PFAS compounds after 24 days of initial dosing with PFAScontaminant solution.

FIG. 14 are bar graphs of the BCFs of Helianthus annuus (Sunflower) forsix target PFAS compounds after 24 days of initial dosing with PFAScontaminant solution.

FIG. 15 are bar graphs of the BCFs of Amaranthus tricolor (Amaranth) forsix target PFAS compounds after 67 days of initial dosing with PFAScontaminant solution.

FIG. 16 are bar graphs of the BCFs of Betula nigra (River Birch) for sixtarget PFAS compounds after 92 days of initial dosing with PFAScontaminant solution.

FIG. 17 are bar graphs of the BCFs of Esquisetum hyemale (Horsetail) forsix target PFAS compounds after 92 days of initial dosing with PFAScontaminant solution.

FIG. 18 are bar graphs of the BCFs of Fraxinus pennsylvanica (Green Ash)for six target PFAS compounds after 92 days of initial dosing with PFAScontaminant solution.

FIG. 19 are bar graphs of the BCFs of Liriodendron tulipifera (TulipPoplar) for six target PFAS compounds after 92 days of initial dosingwith PFAS contaminant solution.

FIG. 20 are bar graphs of the BCFs of Pinus taeda (Loblolly Pine) forsix target PFAS compounds after 92 days of initial dosing with PFAScontaminant solution.

FIG. 21 are bar graphs of the BCFs of Platanus occidentalis (Sycamore)for six target PFAS compounds after 92 days of initial dosing with PFAScontaminant solution.

FIG. 22 is a schematic of a PVC growth chamber (i.e., column) used forresearching and testing plant species and soil management for PFASphytoextraction.

FIG. 23 is a schematic of the randomized three-block study design usedin the Phytoremediation Pilot Study.

In assessing the fitness of plant species to serve in a phytoremediationrole by means of phytoextraction the Bioconcentration Factor (BCF) is akey metric. The BCF is calculated as the plant/soil contaminantconcentration ratio:

BCF_(plant) =C _(plant) /C _(reference media)

A BCF that is >1 is considered to indicate accumulation. For use in aphytoextraction context it is ideal for remedial plants to have a BCF of10 or more. FIGS. 2 through 22 present bar graphs showing the calculatedBCFs for each of six different species based on the successful benchstudy that established “Proof-of-Concept”. These BCFs substantiateefficacy of one or more implementations in accord with the presentconcepts, by way of example only, not by way of limitations.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before explaining the disclosed embodiments of the present invention indetail, it is to be understood that the invention is not limited in itsapplications to the details of the particular arrangements shown sincethe invention is capable of other embodiments. Also, the terminologyused herein is for the purpose of description and not of limitation.

In the Summary above and in the Detailed Description of PreferredEmbodiments and in the accompanying drawings, reference is made toparticular features (including method steps) of the invention. It is tobe understood that the disclosure of the invention in this specificationdoes not include all possible combinations of such particular features.For example, where a particular feature is disclosed in the context of aparticular aspect or embodiment of the invention, that feature can alsobe used, to the extent possible, in combination with and/or in thecontext of other particular aspects and embodiments of the invention,and in the invention generally. In this section, some embodiments of theinvention will be described more fully with reference to theaccompanying drawings, in which preferred embodiments of the inventionare shown.

This invention may, however, be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein.Rather, these embodiments are provided so that this disclosure will bethorough and complete, and will convey the scope of the invention tothose skilled in the art. Like numbers refer to like elementsthroughout, and prime notation is used to indicate similar elements inalternative embodiments.

Other technical advantages may become readily apparent to one ofordinary skill in the art after review of the following figures anddescription.

It should be understood at the outset that, although exemplaryembodiments are illustrated in the figures and described below, theprinciples of the present disclosure may be implemented using any numberof techniques, whether currently known or not. The present disclosureshould in no way be limited to the exemplary implementations andtechniques illustrated in the drawings and described below.

Unless otherwise specifically noted, articles depicted in the drawingsare not necessarily drawn to scale.

Phytoremediation is the direct use of living plants for in situremediation of contaminated soil, sludges, sediments, and groundwaterthrough contaminant removal, degradation, or containment.Phytoextraction, also called phytoaccumulation, refers to the uptake andtranslocation of contaminants in the soil by plant roots into theaboveground portions of the plants. Our study successfully achievedProof-of-Concept for multiple plant species via a bench studyinvestigation signaling the development of an effective phytoremediationprocess and methodology for the cleanup of PFAS in soil, sediment, andgroundwater.

Based on our laboratory data, our Phase I SBIR phytoremediationinnovation has verified hyperaccumulation of multiple PFAS compoundsafter 24 and 98 days of initiating contaminant dosing with the PFAScontaminant solution at multiple contaminant concentrations.Additionally, our work has shown repeatable trends across three plantspecies indicating soil management of salinity has shown a correspondinghigher plant accumulation of PFAS.

Materials and Methods

Based on our knowledge of PFAS, vegetation growing vigorously within thewetted field areas of land treatment sites where PFAS-containingwastewater has historically been applied, and plants known to beefficient at phytoextraction of various contaminants, NAI selected thesixteen species for bench trial pilot study evaluation (Table 1).

TABLE 1 Selected Plant Species for Phase I SBIR Study. Scientific NameCommon Name Amaranthus tricolor Amaranth Betula nigra River BirchBrassica juncea Mustard Cynodon dactylon Bermudagrass Esquisetum hyemaleHorsetail Schedonorus arundinaceus Tall Fescue Festuca rubra Red FescueFraxinus pennsylvanica Green ash Helianthus annuus Sunflower Liquidambarstyraciflua Sweetgum Liriodendron tulipifera Tulip Poplar Trifoliumincarnatum Crimson Clover Pinus taeda Loblolly Pine Philopteridshexagonopter Broad Beech Fern Platanus occidentalis Sycamore Salix nigraBlack Willow

Seedlings were planted in columns containing washed sand. Each columnwas constructed of cut polyvinylchloride (PVC) pipe that was capped atthe bottom and linked by an outlet valve for collection of leachate andcontrol of the water level within the column. The outlet valve tubingwas made of clear silicone so water levels in column could be monitoredvia the clear tubing to assess whether fluid levels in the columns weretoo high or low. Washed sand meeting ASTM C33 gradation standard wasused to reduce contaminant sorption. A sample of the sand was analyzedby the University of Georgia Agricultural and Environmental ServicesSoil, Plant, and Water Laboratory for agricultural parameters. Table 2summarizes the soil test data.

TABLE 2 Agricultural soil test data for the washed sand growth media. %S.U. Base meq/100 g Mehlich 1 mg/kg (ppm) μS/cm pH Saturation CEC Ca FeK Mg Mn Na Ni P Zn Ec 5.91 92.77 0.30 37.70 52.17 6.14 7.15 19.24 3.530.05 1.61 1.07 50

The dimensions of the columns were such that each column was filled withapproximately 6000 cubic centimeters (cm³) of sand which approximatelyequates to approximately 9000 grams (g) at a bulk density ofapproximately 1.5 cubic centimeters per gram (cm3/g) (FIG. 22).

The pilot study was located in a secured greenhouse that was temperaturecontrolled at 25±3° C. and with a relative humidity target range of70±5%. Supplemental lighting was used to extend day length toapproximately 16 hours during the autumn and winter experimental periodsin an attempt to help break dormancy. Experimental units were allocatedin a randomized three-block design. Blocks were physically located todistribute the treatments and replications over the greenhouse microenvironmental conditions. However, block design randomization wascoordinated such that the tree species and herbaceous species wererandomized separately to minimize canopy interference among taller treespecies versus lower growing grasses and forbs (FIG. 23).

The four plant species having the highest salt tolerance (Salix nigra,Liqudambar styraciflua, Festuca rubra, Cynodon dactylon) were alsotreated in separate additional experimental units utilizing a salineirrigant. The saline irrigant was comprised of a solution ofapproximately 2.5 grams per liter (g/L) gypsum (CaSO₄.2H₂O) andapproximately 5 g/L of Epsom salt (MgSO₄.2H₂O) mixed with deionizedwater. This water quality had an approximate electrical conductivity ofapproximately 4.5 decisiemens per centimeter (dS/cm). Thus, a totalnumber of 36 experimental units comprised each block (FIG. 23).

The contaminant dosing solution was a deionized aqueous mix containingapproximately 1 milligram per liter (mg/L) of each of the followingseven compounds:

1) tridecafluorohexane-1-sulfonic acid potassium salt (PFHxS)

2) heptadecafluoro-1-octanesulfonic acid (PFOS)

3) perfluorooctanoic acid (PFOA)

4) perfluoropentanoic acid (PFPeA)

5) undecafluorohexanoic acid (PFHxA)

6) nonafluoro-1-butanesulfonic acid (PFBS)

7) n-methyl perfluorooctane sulfonamide (MeFOSA)

The prepared contaminant solution was sampled using laboratory providedwater sampling vials and a clean pair of nitrile gloves. The sample wasshipped via overnight courier to Eurofins TestAmerica laboratory in WestSacramento, Calif. The laboratory reported concentrations (in units ofnanograms per liter (ng/L) of the analytes as shown in Table 3.

TABLE 3 PFAS concentrations of Dosing Solution as determined bylaboratory analysis. PFAS Compound Analyte Concentration (ng/L) PFPeA1,600,000 PFHxA 2,100,000 PFOA 940,000 PFBS 920,000 PFHxS 890,000 PFOS850,000

Contaminant solution treatments were applied in approximately 100milliliter (mL) doses to the surface of each column using a syringe suchthat the solution was distributed relatively even over the soil surface.Dosing was scheduled weekly. When dosing, staff removed any treatedtextiles or clothing that may have weather, heat, or stain resistantcharacteristics. In addition, dosing applications and samplingactivities were performed after donning a new pair of nitrile gloves.Sampling gloves were exchanged with a new pair at any point the glovesbecame dirty or contact was made with materials or entities that couldhave been a potential cross-contaminant source.

Saline treatment plots were irrigated with approximately 100 mLincrements of saline irrigation water per week or greater if waterlevels in the columns allowed. The columns were fertilized weekly withHoagland complete medium solution (Plant Media Inc.) that supplied plantavailable N, P, K, Ca, Mg, S, B, Fe, Mn, Zn, Cu, and Mo. The Hoagland'ssolution was applied at an application rate of approximately 100 mL of asolution prepared as approximately 1.6 grams of Hoagland's solid mediaper liter of water. The solution was applied to the no-plant controlplots as well.

Initial contaminant dosing and salinity dosing of the plants was tobegin once the plant species exhibited healthy growth. Thephytoremediation experiment was targeted to last 4 months (earlyNovember through March 2019); however, vegetation establishment was slowin many cases due to shortened photoperiod (daylength) and winterdormancy. Dosing began on the herbaceous species on Mar. 8, 2019 and thetree species were first dosed on Mar. 14, 2019. When the initial planttissue sampling took place on Apr. 1, 2019, a total of six doses hadbeen applied to the herbaceous species (i.e., a total of ˜0.6 mg of eachPFAS compound applied to each herbaceous column) and a total of fivedoses had been applied to the tree species (i.e., a total of ˜0.5 mg ofeach PFAS compound applied to each tree column). The last dosing eventwas completed on May 8, 2109; at this time a total of twelve doses hadbeen applied to the herbaceous species (i.e., a total of ˜1.2 mg of eachPFAS compound applied to each herbaceous column) and a total of elevendoses had been applied to the tree species (i.e., a total of ˜1.1 mg ofeach PFAS compound applied to each tree column). Final plant tissuesampling was conducted June 6 through Jun. 14, 2019.

For the tree species, samples of the leaves and petioles were collectedseparately from woody samples of the mainstem and branches. Thus, thelaboratory report for the tree species included separate results forleaves/petioles and woody material. Trifolium incamatum (crimson clover)was only sampled during the initial sampling event as the plantsunderwent senesce prior to the final sampling. In addition, the Fraxinuspennsylvanica (green ash) grew poorly and only three of the six plotsgenerated adequate plant material for collection of leaf/petiolesamples.

The tissue samples were collected using clippers that weredecontaminated between each sample using the following process:

1. Wiping any attached plant matter from the clippers

2. Washing the clippers in a Liquinox® and deionized water solution

3. Rising the clippers with deionized water

4. Rising the clippers with isopropyl alcohol

5. Rising the clippers again with deionized water.

The samples were containerized in plastic baggies and placed in an icedcooler. Prior to shipping the samples were re-packed on fresh ice andshipped via overnight courier with chain-of-custody documentation and asigned custody seal to the Engineering Research Center (ERC) at BrownUniversity School of Engineering in Providence, Rode Island. With eachsample shipment the samples were received in good condition thefollowing morning at the laboratory.

The plant tissue sampling conducted on April 1 occurred only after 24days of the first dosing event due to the initial deadline date of May1, 2019 for responding to the Phase II grant solicitation. Hence, thesampling schedule was moved up to accommodate the 30-day laboratoryanalysis turn-around period. Further, the number of species sampled waslimited to ensure the laboratory could process all the submitted sampleswithin 30 days; additional samples would have potentially jeopardizedreceipt of the sample results in time to respond to the Phase IIsolicitation. The following six species were sampled:

-   -   Salix nigra (Black Willow)    -   Cynodon dactylon (Bermudagrass)    -   Schedonorus arundinaceus (Tall Fescue)    -   Festuca rubra (Red Fescue)    -   Liquidambar styraciflua (Sweetgum)    -   Trifolium incamatum (Crimson Clover)

Due to the plants reaching maturity (i.e., going to seed), the Brassicajuncea (Mustard) and Helianthus annuus (Sunflower) were harvested onApr. 1, 2019 and stored in a laboratory freezer. On May 14, 2019 theAmaranthus tricolor (Amaranth) plants had reached maturity, wereharvested, and stored in the laboratory freezer. The samples stored inthe laboratory freezer were shipped to the analytical laboratory alongwith the final plant samples in June 2019. The final plant tissuesampling was conducted June 6 through Jun. 14, 2019.

Results

From the initial sampling event, a total of 46 samples were analyzed todetermine the dry weight concentration of the six contaminant solutioncompounds: PFOA, PFOS, PFBS, PFPeA, PFHxS, and PFHxA. With the initiallaboratory analyses the laboratory reported the detected presence orabsence of MeFOSA; MeFOSA presence/absence was not reported with thefinal laboratory analyses. The sample extraction procedure utilized wasbased on the procedure by Yoo, et al. (Yoo, H., J. W. Washington, T. M.Jenkins, J. J. Ellington. 2011. Quantitative Determination ofPerfluorochemicals and Fluorotelomer Alcohols in Plants fromBiosolid-Amended Fields using LC/MS/MS and GC/MS. Environmental Scienceand Technology, 45, 7985-7990.). The samples were analyzed using anultra-performance liquid chromatograph (UPLC) equipped with a micro massspectrometer (MS/MS). The sample results are presented in sequentialorder in Table 4. The results are reported on a dry weight basis inunits of micrograms per kilogram (μg/Kg). The results for MeFOSA arereported as either “Present” or “Absent”.

TABLE 4 PFAS Concentrations in Plant Tissue PFPeA PFBS PFHxA PFHxS PFOAPFOS MeFosa Sample Date Plant Treatment (ug/kg) NAI 1LP Jun. 6, 2019River Birch No Treatment 7 <1 10 <1 <1 <1 . . . NAI 1W Jun. 6, 2019River Birch No Treatment <1 <1 <1 <1 <1 <1 . . . NAI 2 Apr. 1, 2019Black Willow Treatment + Saline 726.2 203.3 207.8 284.4 142.7 9.9 AbsentNAI 2LP Jun. 6, 2019 Black Willow Treatment + Saline 19166 260 5569 15332530 277 . . . NAI 2W Jun. 6, 2019 Black Willow Treatment + Saline 12 <141 15 166 5 . . . NAI 3 Apr. 1, 2019 Crimson Clover Treatment 1351.8479.3 2255.8 871.2 767.4 125.8 Absent NAI 4 Apr. 1, 2019 Tall FescueTreatment 1814.2 608.3 3242.5 668 509.2 116.4 Present NAI 4 (100x) Jun.12, 2019 Tall Fescue Treatment 12850 2698 9776 1408 965 290 . . . NAI 6Apr. 1, 2019 Sweetgum Control 0.6 <0.4 2.5 2.7 1.8 <0.4 Absent NAI 6LPJun. 6, 2019 Sweetgum Control 5 <1 17 <1 <1 <1 . . . NAI 6W Jun. 6, 2019Sweetgum Control <1 <1 <1 <1 <1 <1 . . . NAI 7LP Jun. 6, 2019 TulipPoplar Treatment 54996 3798 29777 1521 1036 16 . . . NAI 7W Jun. 6, 2019Tulip Poplar Treatment 3245 46 2014 95 138 11 . . . NAI 9 Apr. 1, 2019Bermudagrass Treatment 1332.9 851.2 3054.4 1187.9 880.4 332.1 PresentNAI 9 (10X) Jun. 12, 2019 Bermudagrass Treatment 4344 1663 4686 572 460223 . . . NAI 10 Apr. 1, 2019 Bermudagrass No Treatment 280 95 237 25 32<1 . . . NAI 10 Jun. 12, 2019 Bermudagrass No Treatment 283.1 204.6952.1 122.7 33.1 <1 Absent NAI 11 Apr. 1, 2019 Sweetgum Treatment 862.280.3 1019.4 18 56.3 <0.4 Absent NAI 11LP Jun. 6, 2019 Sweetgum Treatment248 176 365 277 262 65 . . . NAI 11W Jun. 6, 2019 Sweetgum Treatment2579 10 849 30 320 27 . . . NAI 12 Apr. 1, 2019 Black Willow Treatment373.6 62.3 237.9 166 130.7 4.7 Absent NAI 12LP Jun. 6, 2019 Black WillowTreatment 33475 4097 18411 2749 3406 950 . . . NAI 12W Jun. 6, 2019Black Willow Treatment 370 14 192 37 160 26 . . . NAI 13 Apr. 1, 2019Sunflower No Treatment <1 <1 <1 1 <1 40 . . . NAI 14 Apr. 1, 2019Mustard No Treatment <1 <1 1 <1 <1 <1 . . . NAI 15 Apr. 1, 2019Sunflower No Treatment 538 185 530 281 398 75 . . . NAI 16LP Jun. 6,2019 Loblolly Pine Treatment 1329 55 147 89 105 12 . . . NAI 16W Jun. 6,2019 Loblolly Pine Treatment 2 <1 <1 <1 <1 <1 . . . NAI 17LP Jun. 6,2019 Green Ash No Treatment 55 <1 72 <1 <1 <1 . . . NAI 17W Jun. 6, 2019Green Ash No Treatment 6 <1 4 <1 <1 <1 . . . NAI 18 Apr. 1, 2019 RedFescue Treatment + Saline 1812.5 975.3 3601 2412.1 1857.5 624.9 PresentNAI 18 (100x) Jun. 12, 2019 Red Fescue Treatment + Saline 36796 1387439946 9582 9554 2399 . . . NAI 19 Apr. 1, 2019 Crimson Clover NoTreatment 3.7 <0.4 17 6.5 1.9 1.5 Absent NAI 20 Apr. 1, 2019Bermudagrass Treatment + Saline 1517.8 1282 3064.7 1562.5 811.1 239.4Present NAI 20 (10x) Jun. 12, 2019 Bermudagrass Treatment + Saline 52342249 5410 867 767 267 . . . NAI 21 Apr. 1, 2019 Sweetgum Treatment +Saline 74 86 65.1 42.5 4.6 <0.4 Absent NAI 21LP Jun. 6, 2019 SweetgumTreatment + Saline 193 176 502 946 508 1079 . . . NAI 21W Jun. 6, 2019Sweetgum Treatment + Saline 111 17 67 30 183 191 . . . NAI 22LP Jun. 7,2019 River Birch Treatment 21688 908 16399 2257 2994 870 . . . NAI 22WJun. 7, 2019 River Birch Treatment 1647 4 413 58 135 248 . . . NAI 23Apr. 1, 2019 Red Fescue No Treatment 5.8 6.1 53.3 14.2 5.6 10.5 AbsentNAI 23 Jun. 12, 2019 Red Fescue No Treatment 215 16 143 11 15 <1 . . .NAI 24 Apr. 1, 2019 Red Fescue Treatment 1492 601.2 2738.7 2071.2 1667.4550.4 Present NAI 24 (100x) Jun. 13, 2019 Red Fescue Treatment 180554167 16731 3472 3127 80 . . . NAI 25 Apr. 1, 2019 Tall Fescue NoTreatment 5.5 1.8 19.3 4 4.3 6.4 Absent NAI 25 Jun. 13, 2019 Tall FescueNo Treatment 174 6 145 2 8 <1 . . . NAI 26W Jun. 7, 2019 Green AshTreatment 973 27 338 91 45 6 . . . NAI 27LP Jun. 7, 2019 Tulip PoplarTreatment 60 <1 55 <1 <1 <1 . . . NAI 27W Jun. 7, 2019 Tulip PoplarTreatment 19 <1 4 <1 <1 <1 . . . NAI 28 (100x) May 14, 2019 AmaranthTreatment 47118 253 6347 2593 5551 806 . . . NAI 29 May 14, 2019Amaranth No Treatment 696 1 334 2 14 <1 . . . NAI 30 (100x) Jun. 13,2019 Horsetail Treatment 29235 49 23950 304 1693 114 . . . NAI 31LP Jun.7, 2019 Loblolly Pine No Treatment 5 <1 <1 <1 <1 <1 . . . NAI 31W Jun.7, 2019 Loblolly Pine No Treatment <1 <1 <1 <1 <1 <1 . . . NAI 32LP Jun.7, 2019 Sycamore No Treatment 3 <1 6 <1 <1 <1 . . . NAI 32W Jun. 7, 2019Sycamore No Treatment 1 <1 <1 <1 <1 <1 . . . NAI 33 (100x) Apr. 1, 2019Mustard Treatment 11888 198 4591 714 1413 344 . . . NAI 34 Jun. 13, 2019Horsetail No Treatment 241 <1 294 1 9 <1 . . . NAI 35 Apr. 1, 2019Crimson Clover Treatment 1825.5 844.2 2889.7 459.8 261 0.8 Absent NAI36LP Jun. 7, 2019 Sycamore Treatment 26615 4776 19306 1774 1802 337 . .. NAI 36W Jun. 7, 2019 Sycamore Treatment 71 <1 63 24 91 33 . . . NAI 37Apr. 1, 2019 Black Willow No Treatment 0.8 <0.4 0.6 0.6 0.7 0.5 AbsentNAI 37LP Jun. 7, 2019 Black Willow No Treatment 126 <1 70 <1 <1 <1 . . .NAI 37W Jun. 7, 2019 Black Willow No Treatment 3 <1 1 <1 <1 <1 . . . NAI38 Apr. 1, 2019 Bermudagrass Treatment 1177.2 832.7 2747.3 1168.4 699.5208.5 Present NAI 38 (10x) Jun. 13, 2019 Bermudagrass Treatment 47311886 4404 698 745 241 . . . NAI 39 Apr. 1, 2019 Bermudagrass Treatment +Saline 1315.7 1159.9 2975.8 1328.7 628.9 159.6 Present NAI 39 (10x) Jun.12, 2019 Bermudagrass Treatment + Saline 4914 2127 4290 1068 995 288 . .. NAI 40 Jun. 13, 2019 Horsetail No Treatment 174 5 317 4 13 <1 . . .NAI 41 Apr. 1, 2019 Black Willow No Treatment 0.5 <0.4 <0.4 <0.4 0.5<0.4 Absent NAI 41LP Jun. 7, 2019 Black Willow No Treatment 31 <1 17 <1<1 <1 . . . NAI 41W Jun. 7, 2019 Black Willow No Treatment <1 <1 <1 <1<1 <1 . . . NAI 42W Jun. 7, 2019 Loblolly Pine Treatment 5 <1 7 2 8 3 .. . NAI 42LP Jun. 7, 2019 Loblolly Pine Treatment 880 64 106 111 190 25. . . NAI 44 Apr. 1, 2019 Red Fescue Treatment + Saline 1260.5 529.32030.9 1637.7 1136.7 546.1 Present NAI 44 (100x) Jun. 13, 2019 RedFescue Treatment + Saline 17155 4384 19130 3816 3456 1399 . . . NAI 45May 14, 2019 Amaranth No Treatment 17 <1 93 <1 2 <1 . . . NAI 46LP Jun.7, 2019 Green Ash No Treatment 57 7 59 4 <1 <1 . . . NAI 46W Jun. 7,2019 Green Ash No Treatment 4 <1 1 <1 <1 <1 . . . NAI 47LP Jun. 7, 2019Tulip Poplar Treatment 35075 28428 19919 4428 1743 1620 . . . NAI 47WJun. 7, 2019 Tulip Poplar Treatment 1743 51 1529 58 51 44 . . . NAI 48(10x) Apr. 1, 2019 Sunflower Treatment 6494 191 1963 330 395 90 . . .NAI 49 Apr. 1, 2019 Tall Fescue No Treatment 4.8 3 24.4 5.4 7.3 10.2Absent NAI 49 Jun. 13, 2019 Tall Fescue No Treatment 185 48 179 8 11 2 .. . NAI 50 (100x) May 14, 2019 Amaranth Treatment 64183 324 23866 31857039 754 . . . NAI 51 Apr. 1, 2019 Sweetgum Treatment + Saline 263.696.5 217.7 29.4 7.8 <0.4 Absent NAI 51LP Jun. 7, 2019 SweetgumTreatment + Saline 16645 4355 11189 5393 4534 3840 . . . NAI 51W Jun. 7,2019 Sweetgum Treatment + Saline 388 64 234 24 144 202 . . . NAI 52 Apr.1, 2019 Black Willow Treatment 795.8 229.3 382.6 316 292.9 16.9 AbsentNAI 52LP Jun. 7, 2019 Black Willow Treatment 25191 378 17343 2049 3877413 . . . NAI 52W Jun. 7, 2019 Black Willow Treatment 521 12 248 85 33953 . . . NAI 53 Apr. 1, 2019 Mustard No Treatment <1 <1 <1 <1 <1 <1 . .. NAI 54 Apr. 1, 2019 Tall Fescue Treatment 1011.7 488.3 1906.4 377.6267.5 61.4 Present NAI 54 (10x) Jun. 13, 2019 Tall Fescue Treatment 49712105 4273 870 614 114 . . . NAI 55 Apr. 1, 2019 Sycamore Treatment1390.1 486 2105.3 1490.4 1143.7 432.8 Present NAI 55 (100x) Jun. 13,2019 Sycamore Treatment 25465 5427 21402 5166 4465 1730 . . . NAI 56LPJun. 7, 2019 Sycamore Treatment 11442 143 3212 296 431 46 . . . NAI 56WJun. 7, 2019 Sycamore Treatment 113 5 47 14 46 <1 . . . NAI 57LP Jun. 7,2019 Loblolly Pine No Treatment 14 <1 <1 <1 <1 <1 . . . NAI 57W Jun. 7,2019 Loblolly Pine No Treatment <1 <1 <1 <1 <1 <1 . . . NAI 58 (100x)Apr. 1, 2019 Mustard Treatment 20412 2411 16550 1783 2684 638 . . . NAI59 Apr. 1, 2019 Bermudagrass No Treatment 5.1 <0.4 12.6 1.1 1.4 1.6Absent NAI 59 Jun. 12, 2019 Bermudagrass No Treatment 110 18 78 <1 1 <1. . . NAI 61LP Jun. 10, 2019 River Birch No Treatment 5 <1 17 <1 <1 <1 .. . NAI 61W Jun. 10, 2019 River Birch No Treatment 2 <1 <1 <1 <1 <1 . .. NAI 62 Apr. 1, 2019 Sweetgum No Treatment 1.1 <0.4 2.6 <0.4 0.4 <0.4Absent NAI 62LP Jun. 7, 2019 Sweetgum No Treatment 26 <1 75 <1 <1 <1 . .. NAI 62W Jun. 7, 2019 Sweetgum No Treatment <1 <1 <1 <1 <1 <1 . . . NAI63 Jun. 13, 2019 Red Fescue No Treatment 344 3 191 3 7 <1 . . . NAI 63Apr. 1, 2019 Red Fescue No Treatment 2.5 1.4 44.6 1.8 3.3 2.2 Absent NAI64 (100x) Jun. 14, 2019 Horsetail Treatment 30180 40 19789 276 1263 190. . . NAI 65 Apr. 1, 2019 Sunflower No Treatment <1 6 2 31 11 267 . . .NAI 66W Jun. 1, 2019 Green Ash Treatment 92 187 23 167 22 55 . . . NAI67LP Jun. 7, 2019 Tulip Poplar No Treatment 11 7 12 <1 <1 <1 . . . NAI67W Jun. 7, 2019 Tulip Poplar No Treatment 5 <1 <1 <1 <1 <1 . . . NAI 68Apr. 1, 2019 Crimson Clover No Treatment 5 <0.4 15.7 0.6 0.4 <0.4 AbsentNAI 69 (100x) Jun. 14, 2019 Horsetail Treatment 36682 32 26854 257 1644203 . . . NAI 70 Apr. 1, 2019 Crimson Clover Treatment 1307.7 393.22333.5 413.1 318.3 <0.4 Absent NAI 71 Apr. 1, 2019 Sweetgum Treatment231.6 106.5 196.3 31.4 48.8 <0.4 Absent NAI 71LP Jun. 11, 2019 SweetgumTreatment 5665 414 3047 1977 3226 942 . . . NAI 71W Jun. 11, 2019Sweetgum Treatment 97 6 53 55 336 132 . . . NAI 72LP Jun. 11, 2019Sycamore No Treatment 11 <1 30 <1 <1 <1 . . . NAI 72W Jun. 11, 2019Sycamore No Treatment 3 <1 <1 <1 <1 <1 . . . NAI 73 Jun. 14, 2019 RedFescue No Treatment 86 1 58 <1 1 <1 . . . NAI 73 Apr. 1, 2019 Red FescueNo Treatment 3.1 1.5 8.4 4.3 4.9 5.4 Absent NAI 74 Apr. 1, 2019 TallFescue No Treatment 3.9 1 13 1 2.2 1.4 Absent NAI 74 Jun. 14, 2019 TallFescue No Treatment 231 6 145 2 13 <1 . . . NAI 75 (10x) May 14, 2019Amaranth Treatment 3061 401 10089 2817 4731 348 . . . NAI 76 Apr. 1,2019 Black Willow Treatment + Saline 696.5 102.4 229 160 123.1 <0.4Absent NAI 76LP Jun. 11, 2019 Black Willow Treatment + Saline 25291 45415064 2264 2656 376 . . . NAI 76W Jun. 11, 2019 Black Willow Treatment +Saline 23 <1 32 7 107 <1 . . . NAI 77LP Jun. 11, 2019 River BirchTreatment 32796 1135 23620 3137 5936 1391 . . . NAI 77W Jun. 11, 2019River Birch Treatment 274 4 156 17 159 30 . . . NAI 80 Apr. 1, 2019Mustard No Treatment 14 <1 9 <1 <1 <1 . . . NAI 81LP Jun. 11, 2019 RiverBirch Treatment 31005 1362 20210 3704 7326 3015 . . . NAI 81W Jun. 11,2019 River Birch Treatment 138 <1 91 50 239 263 . . . NAI 82LP Jun. 11,2019 Loblolly Pine Treatment 682 5 25 13 19 <1 . . . NAI 82W Jun. 11,2019 Loblolly Pine Treatment 2 <1 2 <1 <1 <1 . . . NAI 83 Apr. 1, 2019Bermudagrass No Treatment 2.5 <0.4 3.1 1.4 1.4 1.9 Absent NAI 83 Jun.13, 2019 Bermudagrass No Treatment 103 12 72 4 1 <1 . . . NAI 84 (10x)Apr. 1, 2019 Mustard Treatment 6789 944 3944 409 1348 319 . . . NAI 85(10x) Apr. 1, 2019 Sunflower Treatment 4779 159 407 217 291 68 . . . NAI86LP Jun. 11, 2019 Loblolly Pine No Treatment 7 <1 <1 <1 <1 <1 . . . NAI86W Jun. 11, 2019 Loblolly Pine No Treatment <1 <1 <1 <1 <1 <1 . . . NAI87LP Jun. 11, 2019 Sycamore No Treatment 28 <1 26 <1 <1 <1 . . . NAI 87WJun. 11, 2019 Sycamore No Treatment 2 <1 <1 <1 <1 <1 . . . NAI 88 Jun.14, 2019 Horsetail No Treatment 297 3 307 3 17 <1 . . . NAI 89 Apr. 1,2019 Red Fescue Treatment 1121.3 582.2 2156.7 1791.7 1417.1 608.8Present NAI 89 (100x) Jun. 14, 2019 Red Fescue Treatment 22127 982321127 4293 3618 1627 . . . NAI 90 Apr. 1, 2019 Red Fescue Treatment +Saline 2651.4 758.7 2804.6 2148.9 1597.9 719.8 Present NAI 90 (100x)Jun. 14, 2019 Red Fescue Treatment + Saline 19490 3767 16710 3703 31411815 . . . NAI 91LP Jun. 11, 2019 Green Ash Treatment 169 816 690 1353<1 <1 . . . NAI 91W Jun. 11, 2019 Green Ash Treatment 73 14 26 4 1 <1 .. . NAI 92 Apr. 1, 2019 Black Willow Treatment 656.7 136.6 204.6 197.9170.8 5.3 Absent NAI 92LP Jun. 11, 2019 Black Willow Treatment 362715337 21250 2889 3043 306 . . . NAI 92W Jun. 11, 2019 Black WillowTreatment 230 6 117 47 225 17 . . . NAI 93 May 14, 2019 Amaranth NoTreatment 204 2 186 11 15 116 . . . NAI 94 Apr. 1, 2019 BermudagrassTreatment 1145.9 778.1 2736.3 1129.5 840.3 320.3 Present NAI 94 (10x)Jun. 13, 2019 Bermudagrass Treatment 4852 1467 4634 396 559 197 . . .NAI 95 Apr. 1, 2019 Sunflower No Treatment 24 2 11 7 8 52 . . . NAI 96LPJun. 11, 2019 Black Willow No Treatment 43 <1 33 <1 <1 <1 . . . NAI 96WJun. 11, 2019 Black Willow No Treatment 12 <1 3 <1 <1 <1 . . . NAI 97LPJun. 11, 2019 Sycamore Treatment 15456 252 5163 834 1136 404 . . . NAI97W Jun. 11, 2019 Sycamore Treatment 66 1 54 6 52 14 . . . NAI 98 Apr.1, 2019 Crimson Clover No Treatment 2.5 0.6 7 0.4 0.6 <0.4 Absent NAI 99Apr. 1, 2019 Tall Fescue Treatment 1015 492.6 1777.3 433.8 367.9 60.6Present NAI 99 (100x) Jun. 14, 2019 Tall Fescue Treatment 26518 937123988 2769 1722 388 . . . NAI100 Apr. 1, 2019 Bermudagrass Treatment +Saline 1302.3 1142.2 2234.6 1545.6 985.6 263.2 Present NAI 100 (10x)Jun. 13, 2019 Bermudagrass Treatment + Saline 4804 1564 4164 622 690 286. . . NAI101 Apr. 1, 2019 Sweetgum Treatment 1254.4 142.6 1311.5 145.4175.3 <0.4 Absent NAI 101LP Jun. 11, 2019 Sweetgum Treatment 298 335 531557 501 168 . . . NAI 101W Jun. 11, 2019 Sweetgum Treatment 268 6 147 37406 56 . . . NAI 102LP Jun. 11, 2019 Tulip Poplar Treatment 17853 184074119 3032 1366 805 . . . NAI 102W Jun. 11, 2019 Tulip Poplar Treatment189 8 233 15 22 31 . . . NAI 103LP Jun. 12, 2019 Black WillowTreatment + Saline 39416 544 15533 4999 4771 2670 . . . NAI 103W Jun.12, 2019 Black Willow Treatment + Saline 177 2 100 97 381 180 . . . NAI104LP Jun. 12, 2019 River Birch No Treatment 79 12 71 28 76 6 . . . NAI104W Jun. 12, 2019 River Birch No Treatment <1 <1 <1 <1 <1 <1 . . .NAI105 Apr. 1, 2019 Sweetgum Treatment + Saline 415.7 65.9 705.1 70.469.9 <0.4 Absent NAI 105LP Jun. 12, 2019 Sweetgum Treatment + Saline4064 386 2720 2239 2372 359 . . . NAI 105W Jun. 12, 2019 SweetgumTreatment + Saline 208 10 155 39 310 101 . . . NAI 106W Jun. 12, 2019Green Ash No Treatment 2 <1 <1 <1 <1 <1 . . . NAI 107LP Jun. 12, 2019Tulip Poplar No Treatment 7 <1 4 <1 <1 <1 . . . NAI 107W Jun. 12, 2019Tulip Poplar No Treatment 4 <1 <1 <1 <1 <1 . . . NAI108 Apr. 1, 2019Sweetgum No treatment 1.1 <0.4 2.6 <0.4 0.7 <0.4 Absent NAI 108LP Jun.12, 2019 Sweetgum No treatment 15 <1 50 <1 <1 <1 . . . NAI 108W Jun. 12,2019 Sweetgum No treatment <1 <1 <1 <1 <1 <1 . . .

Table 5 presents the average final sampling results by species andtreatment (except crimson clover, mustard, and sunflower which were allsampled only once at the initial sampling event). Of the herbaceousspecies, the highest average plant tissue concentrations were observedin Festuca rubra (Red Fescue) that received contaminant treatments withsaline irrigant. An exception is for PFOA, where Amaranthus (Amaranth)showed the highest average concentration of PFOA. Of the tree species,the highest average plant tissue concentrations were observed in Betulanigra-LP (River Birch-LP), except for PFBS where Liriodendrontulipifera-LP (Tulip Poplar-LP) showed the highest average. Along withBetula nigra-LP and Liriodendron tulipifera-LP, Liquidambarstyraciflua-LP (Sweetgum-LP) and Salix nigra-LP (Black Willow-LP) showedhigher overall accumulations of PFAS compounds, though at slightly loweraverages.

The Cynodon dactylon (Bermudagrass) control column NAI 10, wasinadvertently dosed on March 22 with approximately 70 mL of contaminantsolution. The data from NAI 10 was not utilized in calculating averagevalues presented in Table 5.

TABLE 5 Average PFAS concentrations in final plant tissue samples foreach species and treatment. PFBS PFPeA PFHxA PFHxS PFOA PFOS Plant TypeTreatment (μg/Kg) (μg/Kg) (μg/Kg) (μg/Kg) (μg/Kg) (μg/Kg) LiquidambarControl <1 15.3 47.3 <1 <1 <1 styraciflua-LP Treatment 308.3 2070.31314.3 937.0 1329.7 391.7 Treatment + Saline 1639.0 6967.3 4803.7 2859.32471.3 719.0 Liquidambar Control <1 <1 <1 <1 <1 <1 styraciflua-WTreatment 7.3 981.3 349.7 40.7 354.0 71.7 Treatment + Saline 30.3 235.7152.0 31.0 212.3 164.7 Salix nigra-LP Control 0.0 66.7 40.0 <1 <1 <1Treatment 3270.7 31645.7 19001.3 2562.3 3442.0 556.3 Treatment + Saline419.3 27957.7 12055.3 2932.0 3319.0 1107.7 Salix nigra-W Control <1 5.01.3 <1 <1 <1 Treatment 10.7 373.7 185.7 56.3 241.3 32.0 Treatment +Saline 0.7 70.7 57.7 39.7 218.0 61.7 Liriodendron Control 2.3 26.0 23.7<1 <1 <1 tulipifera-LP Treatment 16877.7 35974.7 17938.3 2993.7 1381.7813.7 Liriodendron Control 29.5 650.3 588.7 36.5 36.5 37.5 tulipifera-WTreatment 46.0 1084.7 2014.0 95.0 138.0 11.0 Plantanus Control 0.0 14.020.7 0.0 0.0 0.0 occidentalis-LP Treatment 1723.7 17837.7 9227.0 968.01123.0 262.3 Plantanus Control <1 2.0 <1 <1 <1 <1 occidentalis-WTreatment 2.0 83.3 54.7 14.7 63.0 15.7 Pinus taeda-LP Control <1 8.7 <1<1 <1 <1 Treatment 41.3 963.7 92.7 71.0 104.7 12.3 Pinus taeda-W Control<1 <1 <1 <1 <1 <1 Treatment <1 3.0 3.0 0.7 2.7 1.0 Fraxinus Control 3.556 65.5 2.0 <1 <1 pennsylvanica-LP Treatment 816.0 169.0 690.0 1353.0 <1<1 Fraxinus Control <1 4.0 1.7 <1 <1 <1 Pennsylvania-W Treatment 76.0379.3 129.0 87.3 22.7 20.3 Betula nigra-LP Control 4.0 30.3 32.7 9.325.3 2.0 Treatment 1135.0 28496.3 20076.3 3032.7 5418.7 1758.7 Betulanigra-W Control <1 0.7 <1 <1 <1 <1 Treatment 2.7 686.3 220.0 41.7 177.7180.3 Festuca Rubra Control 6.7 215.0 130.7 4.7 7.7 0.0 Treatment 6472.321882.3 19753.3 4310.3 3736.7 1145.7 Treatment + Saline 7341.7 24480.325262.0 5700.3 5383.7 1871.0 Cynodon Control 15.0 75.0 1.0 2.0 1.0 <1Dactylon Treatment 1672.0 4574.7 588.0 555.3 588.0 220.3 Treatment +Saline 1980.0 4621.3 817.3 852.3 817.3 280.3 Trifolium Control 3.7 0.613.2 2.5 1.0 0.5 incarnatum* Treatment 1495.0 572.2 2493.0 581.4 448.942.2 Schedonorus Control 20.0 196.7 156.3 4.0 10.7 0.7 arundinaceusTreatment 4724.7 14779.7 12679.0 1682.3 1100.3 264.0 Helianthus annusControl 4.0 24.0 6.5 13.0 9.5 119.7 Treatment 178.3 3937.0 966.7 276.0361.3 77.7 Brassica juncea Control <1 4.7 3.0 <1 <1 <1 Treatment 1184.313029.7 8361.7 968.7 1815.0 433.7 Amaranthus Control 1.5 305.7 204.3 6.510.3 38.7 tricolor Treatment 326.0 38120.7 13434.0 2865.0 5773.7 636.0Equisetum Control 4.0 237.3 306.0 2.7 13.0 0.0 hyemale Treatment 40.332032.3 23531.0 279.0 1533.3 169.0

In assessing the fitness of plant species to serve in a phytoremediationrole by means of phytoextraction, the Bioconcentration Factor (BCF) is akey metric. The BCF is calculated as the plant/soil contaminantconcentration ratio:

BCF_(plant) =C _(plant) /C _(reference media)

A BCF that is >1 is considered to indicate accumulation. However, foruse in a phytoextraction context, it is ideal for remedial plants tohave a BCF of 10 or more. An additional criterion of note is when aplant accumulates an amount of a substance (i.e., contaminant) that istwo orders of magnitude greater accumulation than that found in plantsgrowing in uncontaminated media. A plant meeting these criteria isconsidered a hyperaccumulator (McCutcheon, et al., 2003).

BCFs were calculated for each contaminant for all species. The soil PFASconcentrations were calculated based on the measured volumes of soil inthe columns and the amount of contaminant solution dosed. In calculatingthe soil concentrations, the nominal concentrations of PFAS compoundsdosed were adjusted per the dosing solution laboratory results (Table3). FIGS. 2 through 22 present bar graphs showing the calculated BCFsfor each herbaceous species and each tree species (leaf-petiole tissuesamples) for each contaminant.

With respect to the higher profile PFAS compounds (PFOS, PFOA, PFBS) thefollowing species showed BCF concentrations >10 for one or more: FestucaRubra (Red Fescue), Betula nigra-LP (River Birch-LP), Liquidambarstyraciflua-LP (Sweetgum-LP), Salix nigra-LP (Black Willow-LP),Plantanus occidentalis-LP (Sycamore-LP), Liriodendron tulipifera-LP(Tulip Poplar-LP), Amaranthus tricolor (Amaranth), Schedonorusarundinaceus (Tall Fescue), Cynodon Dactylon (Bermudagrass), Brassicajuncea (Mustard), Equisetum hyemale (Horsetail). With respect to thetree species, hyperaccumulation was observed in the leaves and petiolesand not in the woody mass (mainstems and branches). The BCFs for thesecompounds are summarized in Table 6.

TABLE 6 Species exhibiting BCFs greater than 10. BCF BCF Saline BCFSpecies Compound Treatment Treatment Control River Birch - LP PFOS 16.4— 0 Red Fescue 11 17.9 0 Sweetgum - LP 3.6 16   0 Black Willow - LP 5.210.3 0 Amaranth PFOA 45 — 0.1 Horsetail 12.1 — 0.1 Mustard 14.1 — 0River Birch - LP 45.8 — 0.2 Sweetgum - LP 10.9 20.3 0 Red Fescue 32.346.5 0.1 Black Willow - LP 28.9 27.9 0 Tulip Poplar - LP 11.5 — 0Sweetgum - LP PFBS 2.6 13.8 0 Black Willow - LP 28.1  3.6 0 Bermudagrass14 16.5 0.1 Red Fescue 57.2 64.9 0.1 Sycamore - LP 15.2 — 0 TulipPoplar - LP 143.7 — 0 Tall Fescue 39.6 — 0.2 — = Treatment Not Tested LP= leaves and petioles

Discussion

Our results establish Proof-of-Concept for phytoremediation of multiplePFAS compounds. Festuca rubra (Red Fescue) preformed the best overall atcontaminant accumulation and achieved hyperaccumulation of all six PFAScompounds including PFOA, PFOS, and PFBS over the course of the study(as well as after 24 days from initial contaminant dosing when the firstsampling event was completed on Apr. 1, 2018) in both the contaminanttreatment and the contaminant+salinity treatment. The average red fescueBCF of the contaminant+salinity treatment showed greater accumulationthan the treatment only for all six PFAS compounds. The finalcontaminant+salinity treatment samples had BCFs that ranged fromapproximately 17.9 (PFOS) to approximately 124.4 (PFPeA). Thecontaminant treatment had BCFs that ranged from approximately 11.0(PFOS) to approximately 111.2 (PFPeA).

The large difference in PFAS accumulation between red fescue (Festucarubra) compared to tall fescue (Schedonorus arundinaceus) is notable.However, there has been controversy regarding the naming andclassification of the fescues (Casler, et al. 2008). The fine fescues,such as red fescue, are classified in the genus Festuca. The broadleaffescues, such as tall fescue, were originally grouped into Festuca. Asthe science of plant classification developed and became moresophisticated, the broadleaf fescues were given their own genus:Schedonorus (Casler, et al. 2008). Hence, differences in accumulation ofPFAS coincide with significant differences with the two plant species.

In addition to the red fescue, the leaves and petioles associated withthe contaminant+salinity treatment for Liquidambar styraciflua(Sweetgum-LP) achieved hyperaccumulation of all six PFAS compounds. Thecontaminant+salinity treatment samples had BCFs that ranged fromapproximately 13.8 (PFBS) to approximately 33.7 (PFPeA). The woodysamples showed negligible PFAS concentrations.

As a species, the black willow leaves and petioles achievedhyperaccumulation of all six PFAS compounds, but not within the sametreatment. Black willow leaves and petioles achieved hyperaccumulationfor the contaminant+salinity treatment for all but PFBS, whereas, theyachieved hyperaccumulation for the contaminant only treatment for allbut PFOS. The woody samples showed negligible PFAS concentrations.

River birch leaves and petioles showed hyperaccumulation for all butPFBS, but the BCF for PBFS was approximately 9.8. River birch leaves andpetioles had the highest BCF for PFOA in study with a value ofapproximately 45.8. The woody samples showed negligible PFASconcentrations.

Like river birch, tulip poplar leaves and petioles showedhyperaccumulation for all but PFOS, but the BCF for PFOS wasapproximately 7.5. Tulip poplar leaves and petioles had the highest BCFfor any of the tested PFAS compounds with a value of approximately 176.1for PFPeA. The woody samples showed negligible PFAS concentrations.

Although Amaranthus did not achieve hyperaccumulation for PFBS or PFOS,it did hyperaccumulate the remaining four PFAS compounds and had one ofthe highest BCFs for PFOA (approximately 45.0).

Given Festuca rubra is shade tolerant and its growing seasons are springand fall (i.e., cool season grass), its use with a successfullyhyperaccumulating deciduous tree species is suited to work in tandem.This approach would involve a double-canopy system or a double croppingsystem. A double-canopy system entails management of two remedial crops,whereby the cool season red fescue grass grows during the fall andspring and a deciduous tree species (e.g., willow, sweetgum, riverbirch, tulip poplar) is grown to a degree of maturity. In this scenariothe trees break winter dormancy and begin to leaf-out just as the growthof the red fescue crop peaks, is harvested, and then grass growthdeclines as summer temperatures increase.

A double cropping system using red fescue and a deciduous tree speciesentails a somewhat more innovative approach. This approach uses elementsof an intensive forest management technique of short or ultrashortrotation coppice (SRC). The SRC silviculture practice contemplated inthis approach involves annual harvests using a system principallyassociated with maximizing woody biomass production as a bioenergy crop(Hauk, et al., 2014). Early development of short-rotation forestmanagement techniques and systems were initially developed to a thoroughdegree in the 1970s when an energy crisis and pulp & paper demandsnecessitated innovations to meet national needs and industrial demands(Steinbeck, 1972; EPA, 1978). These management systems have been furtherrefined since, and SRC has experienced renewed focus as concerns andcosts associated with fossil fuel and climate change have intensified(Hauk, et al., 2014). With these systems both red fescue and the annualgrowth of managed saplings are both mechanically harvested. UltrashortSRC, where hardwoods are harvested annually with power scythes, has beenreferred to as “wood grass”; it has been shown to be technologically andeconomically feasible (Kopp, et al., 1993). Hence, the use of coolseason red fescue in combination with wood grass has merit. With such anapproach red fescue would be harvested as the tree crop emerges fromdormancy and the hardwood “wood grass” would be harvested in late summeror early autumn before leaf fall. With SRC, hardwoods regenerate eachyear from coppice and the need for replanting to replace mortality ismanageable, even with annual harvests (e.g., <30% to 50% mortality afterfive successive annual harvests) (Kopp, et al., 1993). Vigorousregeneration from coppice stools can often persist beyond 30 years(Kopp, et al., 1993).

In this study, the growth media consisted of low ionic exchange screenedsand with little organic matter. This growth media generated conditionswhere the PFAS compounds were more readily available for plant uptakethan would be found in a typical field situation. However, the rationalesupporting the use of a salinity treatment is that it should increaseplant uptake of PFAS, which is supported by our results, because asaline solution, such as one containing divalent calcium and magnesiumsalts, creates greater ionic strength within the soil solution and helpsdisplace PFAS compounds bound to soil exchange sites and organic matter.This increases the PFAS compounds in the soil solution, rendering themmore available for plant uptake.

We note that our target soil salinity was approximately 4,000 pS/cm,which is the level that defines a saline soil condition. However, thesaline-treated plants utilized are reportedly tolerant of this salinitylevel. The salinity level was raised incrementally during the study withregular doses of the saline treatment. The rate of increase in soilsalinity levels was more gradual and slower than anticipated. During thestudy, soil conductivity measurements were made using a direct soilconductivity meter. At the time of the final plant sampling, the controland treatment soils had soil electrical conductivity levels ofapproximately 50 to approximately 100 pS/cm; the treatment+saline soilshad an average conductivity of approximately 1,220 pS/cm. Thus, withfurther increases in soil salinity greater increases in PFAS uptake bythe plants may occur than achieved in this study.

The screened and washed sand used as the growth media in our study waslow in organic matter; although, the grasses and legumes that were usedin the study were first propagated from seed in shallow trays of pottingsoil which contributed some organic matter content when these plantswere transplanted into the columns. Nevertheless, the plants were grownin a relatively low organic matter medium. We understand the plantsuptake PFAS with uptake of soil-water. If impacted sites are managedsuch that organic matter and soluble carbon are reduced, then aconcomitant increase in uptake rates of PFAS by plants will occur. Sitemanagement that includes elements of tillage and application ofsynthetic inorganic nitrogen fertilizer are strategies to lower soilcarbon levels and increase PFAS phytoextraction. When combined withsalinity treatments, some of the negative effects of reduced soilorganic matter, particularly in loamy or clayey soils, can be lessenedby the beneficial flocculating effect of the divalent cations calciumand magnesium.

The higher concentrations of PFAS in the forage grass is also understoodto be partially related to the protein content in the plant tissue, assome researchers exploring PFAS prevalence in agricultural crops havefound higher PFAS concentrations correlating positively with higherprotein contents. Harvesting younger growth on a more frequent harvestschedule, like that which drives greater nitrogen assimilation onnitrogen-limiting land treatment sites, is a strategy applicable todriving increased PFAS assimilation within a phytoremediation context.

Festuca species are cool season grasses that are best planted in theautumn and harvested in spring, before deciduous trees fully leaf out.Festuca can be propagated from seed and yields can typically meetamounts of approximately 3 tons per acre or more. With ultrashort SRCsystems yields can average approximately 10 oven-dry tons per hectareper year (o.d. t ha⁻¹y⁻¹) with fertilization. For example, one studyreported production capacities of various willow tree species rangedfrom approximately 8.0 to approximately 15.5 o.d. t ha⁻¹yr⁻¹ over amultiyear study (Kopp, et al., 1993). At such rates of agronomic/forestproduction, along with the ability to mechanically cultivate andharvest, the viability of phytoextraction of PFAS from contaminated soiland sediment is practical and a “greener cleanup” technology in that itmeets core elements outlined in the American Society of TestingMaterials (ASTM) E2893 Standard Guide for Greener Cleanups that includeminimizing greenhouse gas emissions, air pollutants, use of materials,generation of waste, disturbance to land and ecosystems, and noise andlight disturbance. This is particularly the case on sites where PFASconcentrations are at moderate to lower levels and where the size of theimpacted area is large (>1 acre).

As an example, a cubic foot of soil that is similar to the tested growthmedia and having concentrations of approximately 110 ug/Kg (ppb) of PFOSand PFOA that undergoes phytoremediation with red fescue and utilizingsalinity enhancement, it is estimated it would take approximately 10years and approximately 3.5 years to reduce the concentrations of PFOSand PFOA by approximately 50%, respectively. However, if red fescue isutilized in tandem in a double cropping system, this time frame couldpotentially be reduced by approximately 30%.

Overall, our results demonstrate the feasibility of remediating PFAScontaminated sites. We seek to protect our viable intellectual propertyvia patent protections.

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The term “approximately”/“approximate” can be +/−10% of the amountreferenced. Additionally, preferred amounts and ranges can include theamounts and ranges referenced without the prefix of being approximately.

Although specific advantages have been enumerated above, variousembodiments may include some, none, or all of the enumerated advantages.

Modifications, additions, or omissions may be made to the systems,apparatuses, and methods described herein without departing from thescope of the disclosure. For example, the components of the systems andapparatuses may be integrated or separated. Moreover, the operations ofthe systems and apparatuses disclosed herein may be performed by more,fewer, or other components and the methods described may include more,fewer, or other steps. Additionally, steps may be performed in anysuitable order. As used in this document, “each” refers to each memberof a set or each member of a subset of a set.

To aid the Patent Office and any readers of any patent issued on thisapplication in interpreting the claims appended hereto, applicants wishto note that they do not intend any of the appended claims or claimelements to invoke 35 U.S.C. 112(f) unless the words “means for” or“step for” are explicitly used in the particular claim.

While the invention has been described, disclosed, illustrated and shownin various terms of certain embodiments or modifications which it haspresumed in practice, the scope of the invention is not intended to be,nor should it be deemed to be, limited thereby and such othermodifications or embodiments as may be suggested by the teachings hereinare particularly reserved especially as they fall within the breadth andscope of the claims here appended.

We claim:
 1. A method for increasing the amount of per- andpolyfluoroalkyl substances (PFAS) that a plant will accumulate from PFAScontaminated media, soil, sediment, and groundwater, comprising thesteps of: growing live selected plants in per- and polyfluoroalkylsubstances (PFAS) contaminated media, soil, sediments, or groundwater;and providing for phytoremediation via phytoextraction of the per- andpolyfluoroalkyl substances (PFAS) from the contaminated media, soil,sediments or the groundwater, wherein the providing step furtherincludes managing soil salinity levels to increase ionic strength andsalinity of the contaminated media, soil, sediments and ground waterwith CaSO₄.2H₂O to increase the ionic strength and salinity of thecontaminated media to have a salinity of approximately 1,220 pS/cm.
 2. Amethod for increasing the amount of per- and polyfluoroalkyl substances(PFAS) that a plant will accumulate from PFAS contaminated media, soil,sediment, and groundwater, comprising the steps of: growing liveselected plants in per- and polyfluoroalkyl substances (PFAS)contaminated media, soil, sediments, or groundwater; and providing forphytoremediation via phytoextraction of the per- and polyfluoroalkylsubstances (PFAS) from the contaminated media, soil, sediments or thegroundwater, wherein the providing step further includes managing soilsalinity levels to increase ionic strength and salinity of thecontaminated media, soil, sediments and ground water with MgSO₄.2H₂O toincrease the ionic strength and salinity of the contaminated media tohave a salinity of approximately 1,220 μS/cm.
 3. A method for increasingthe amount of per- and polyfluoroalkyl substances (PFAS) that a plantwill accumulate from PFAS contaminated media, soil, sediment, andgroundwater, comprising the steps of: growing live selected plants inper- and polyfluoroalkyl substances (PFAS) contaminated media, soil,sediments, or groundwater; and providing for phytoremediation viaphytoextraction of the per- and polyfluoroalkyl substances (PFAS) fromthe contaminated media, soil, sediments or the groundwater, wherein theproviding step further includes managing soil salinity levels toincrease ionic strength and salinity of the contaminated media, soil,sediments and ground water with MgSO₄.2H₂O to increase the ionicstrength and salinity of the contaminated media to have a salinity ofapproximately 4,000 μS/cm.